Photo-activatable fluorophores (caged dyes) have wide applications in tracking the spatiotemporal dynamics of molecular movements in biological systems. Previously developed caged fluorophores were designed for photolysis using ultra-violet (UV) light. To exploit the excellent three-dimensional (3D) selectivity of two photon (2P) excitation, new caged fluorophores and techniques suitable for 2P uncaging and imaging need to be developed. The long-term objective of this project is to develop caged dyes and techniques for biological imaging applications. The proposed project will focus on developing new methods to track molecular movements between cells through gap junction (GJ) channels. Using photo-activatable fluorophores to image cell-cell communication represents a new application of caged fluorophores. We aim to achieve three goals: first, to develop a 2P uncaging and imaging technique using cell permeable and caged fluorophores to study the dynamics of cell coupling in physiological preparations consisting of intact cell populations in three dimension;second, to develop 2P activatable, water soluble, and amine reactive fluorophores for bioconjugations. We plan to prepare two classes of bioconjugates (Type 1 and Type 2) that have distinct biological applications;third, to apply these bioconjugates of caged fluorophores to develop 2P uncaging and imaging techniques to trace cell lineage and to monitor GJ intercellular communication (GJIC) in living model organisms such as the nematode C.elegans. This organism is ideal for optical imaging because of its transparency and small size. The proposed research will combine organic synthesis, photochemistry, cell biology, and two photon laser scanning microscopy (2PLSM) to achieve these goals. Although the biological focus of this developmental project is on GJIC, concepts, techniques, and tools developed here should be of general utilities to study both intra- and intercellular molecular dynamics in a variety of biological systems. Abnormal cell-cell communication has been implicated in a number of diseases including cardiac arrhythmia, deafness, neuronal demyelination, and cataracts, developing new techniques to study cell communication is important for devising new strategies for treating these diseases.